This invention was made with Government support under National Institutes of Health Grant No. AR40661. The Government has certain rights in the invention.
(1) Field of the Invention
The present invention generally relates to the modulation of enzyme activity in a cell or animal. More specifically, the invention relates to enhancement of the inactivation of matrix metalloproteinase activity and uses thereof in treating disorders mediated by the matrix metalloproteinase.
(2) Description of the Related Art
Matrix metalloproteinases are enzymes involved in numerous biological functions such as, for example, extracellular matrix remodeling during development, wound healing, and in certain disease processes (Partridge et al., 1996, Crit. Rev. Eukar. Gene Expression 6:15; Stahle-Backdahl et al., 1997, Lab. Invest. 76:717). The matrix metalloproteinase family includes collagenase-3 (MMP-13), interstitial collagenase (MMP-1), PMN collagenase (MMP-8), gelatinases (MMP-2, MMP-9, MMP-7), and stromelysins (MMP-3, MMP-10, MMP-11). These enzymes all share a similar domain structure, have an inactive proenzyme form as synthesized, function at neutral pH, and require zinc and calcium ions for activity. The collagenases are composed of three domains: an N-terminal propeptide domain, a catalytic domain, and a C-terminal hemopexin domain.
Collagenase-3 (MMP-13), one of the matrix metalloproteinases noted above, was originally cloned from a metastatic breast carcinoma cell line and has been associated with that disease (Freije et al., 1994, J. Biol. Chem. 269:16766). The enzyme is also expressed in cartilage and bone. Collagenase-3 acts at physiological pH to degrade fibrillar native collagens (types I, II, and III) and aggrecan (Fosang et al., 1996, FEBS Let. 80:7). Degradation of type II collagen in cartilage explant cultures has been correlated with MMP activity (Kozaci et al., 1997, Arth. Rheumatism. 40:164).
A specific endocytotic receptor system for collagenase-3 has been shown to occur on osteoblastic cells. Two receptors are required for endocytosis. One receptor (the collagenase-3 receptor) is a 170 kDa protein and another is a 600 kDa protein identified as the low density lipoprotein receptor-related protein (Walling et al., 1998, Bone 23:S326. The collagenase-3 receptor is now known to be a novel member of the macrophage mannose receptor type C lectin family. That receptor was first isolated and cloned by Wu et al., 1996, J. Biol. Chem. 271:21323, although it was not recognized at that time as the collagenase-3 receptor.
In the receptor-mediated endocytosis process, collagenase-3 first binds the collagenase-3 receptor. This enzyme-receptor complex then interacts with low density lipoprotein receptor-related protein, after which collagenase-3 is internalized, processed (through endosomes, the trans-Golgi network, and lysosomes), and excreted from the cell in degraded form (Walling et al., 1998, J. Cellular Physiol. 177:563).
Excessive activity of matrix metalloproteinases such as collagenase-3 has been associated with certain diseases, for example osteoarthritis. Osteoarthritis, the most common form of joint disease, results in a slowly progressive degeneration of articular cartilage, particularly at the weight-bearing joints and fingers. Nearly 10% of adults over age 35 are afflicted with osteoarthritis, and the prevalence approaches 85% by age 75 (Felson et al., 1998, Arthritis and Rheumatism 41:1343). The disease is characterized radiologically by narrowing of the joint space (due initially to loss of articular cartilage), increased bone density (secondary to subchondral sclerosis), and osteophyte development. Pain and restricted motion accompany joint destruction. At present, there is no curative treatment for osteoarthritis, and supportive measures and joint replacement surgery are the only options.
Though the pathogenesis is multifactorial, a common endpoint of osteoarthritis is destruction of the cartilage matrix, and mounting evidence suggests that a pathophysiological catalyst for osteoarthritis is a disruption of the normal balance of cartilage synthesis and degradation (McAnulty et al. Pp 140-142 In: Kuettner A, ed. Methods in Cartilage Research. Academic Press Inc, San Diego). The principal components of the fibrillar meshwork, type II collagen and aggrecan, are eroded in osteoarthritis (Hollander et al., 1995, J. Clin. Invest. 96:2859; Lohmander et al., 1995, J. Rheumatol. 22(Suppl 43):75). Osteoarthritic chondrocytes demonstrate increased production of proteoglycans but have a limited capacity to generate new cartilage (Aigner et al., 1997, Arthritis and Rheumatism 40:562), suggesting an imbalanced repair response (Cs-Szabo et al., 1997, Arthritis and Rheumatism, 40:1037). Moreover, compromise of the collagen network leads to irreversible cartilage destruction (Shingleton et al., 1996 Biochem. Cell Biol. 74:759.
Several clinical studies have reported increased levels of collagenase-3 in osteoarthritic synovial fluid (Pelletier et al., 1983, Arthritis and Rheumatism 26:63). Compared to normal chondrocytes, collagenase-3 is overexpressed by arthritic chondrocytes (Reboul et al., 1996, J. Clin. Invest. 97:2011); it is expressed by osteoarthritic synoviocytes but is undetectable in normal synovia (Martel-Pelletier et al., 1994, Lab. Investig. 70:807; Wernicke et al., 1996, J. Rheumatol. 23:590). Matrix degradation products released to synovial fluid may induce exacerbating inflammation (Lohmander et al., 1997, J. Rheumatol. 24:782). Collagenase-3 receptor dysfunction has been implicated in the etiology of osteoarthritis (Irvine et al., Nov. 16, 1998, 2nd Symposium International Cartilage Repair Society; Walling et al., Apr. 24, 1999, Research Poster Finalist #19, SCP-ASIM Medical Student Competition.
Other matrix metalloproteinases have also been associated with human disease. For example, stromelysin-1 (MMP-3) is associated with arthritis and tumor invasion (Becker et al., 1995, Protein Sci. 4:1966), gelatinase A (MMP-2) is associated with cancer metastasis, interstitial collagenase (MMP-1) is associated with Werner""s syndrome (Bauer et al., 1986, Science 234:1240), stromelysin-2 (MMP-10) is associated with cancer (Muller et al., 1988, Biochem. J. 253:187), stromelysin-3 (MMP-11) is associated with breast cancer, and gelatinase B (MMP-9) is associated with osteoarthritis (Fujisawa et al., 1999, J. Biochem. 125:966).
Because of the importance of matrix metalloproteinases in general and collagenase-3 in particular, there is a need for improved methods of regulating these enzymes. The apparent role of collagenase-3 in osteoarthritis and possibly other diseases where degradation of collagen may be involved (such as other arthritic diseases, osteoporosis and post-surgical osteolysis [i.e., aseptic loosening of implants]) makes the need for methods of reducing collagenase-3 activity particularly acute.
Accordingly, the inventor has succeeded in discovering that matrix metalloproteinases are inactivated by endocytosis and degradation. Surprisingly, this process can be increased by various agents, in particular HMG-CoA reductase inhibitors, also known as statins. These agents can be applied in the treatment of diseases which are mediated by an excessive amount of a matrix metalloproteinase.
Thus, one embodiment of the present invention is directed to a method for inactivating a matrix metalloproteinase in a vertebrate cell. The method comprises administering to the cell an effective amount of an agent which causes an increase of endocytosis of the matrix metalloproteinase. Preferably, endocytosis of the matrix metalloproteinase is increased by increasing the activity of low density lipoprotein receptor-related protein. In particularly preferred embodiments, mammalian cells are treated to increase endocytosis of collagenase-3. The preferred agents are HMG-CoA reductase inhibitors, for example pravastatin, atorvastatin, or lovastatin.
The present invention is also directed to a method for treating a vertebrate with a disorder mediated by collagenase-3 activity. The method comprises administering to the vertebrate an effective amount of an agent which increases endocytosis of collagenase-3. Preferably, the agent increases low density lipoprotein receptor-related protein activity, most preferably by increasing expression of the protein. Preferred agents are HMG-CoA reductase inhibitors. The method is particularly useful for mammals where the disorder is arthritis, breast cancer, osteoporosis, or post-surgical osteolysis, most preferably osteoarthritis. In another embodiment, the method further comprises selecting the agent by testing candidate agents for activity in increasing endocytosis of collagenase-3 mediated by low density lipoprotein receptor-related protein.
In another embodiment, the present invention is directed to an assay for determining whether an agent is effective in treating a disorder mediated by collagenase-3. The assay comprises testing the agent for activity in increasing endocytosis of collagenase-3 in a vertebrate cell. The agent is preferably tested by determining levels of excretion of degraded collagenase-3 before and after treatment of the cell with the agent. The assay preferably utilizes mammalian osteoblasts, chondrocytes, or synoviocytes. In one preferred embodiment, the assay utilizes a chondrocyte or synoviocyte to treat osteoarthritis. In another preferred embodiment, the assay utilizes an osteoblast to treat osteoporosis or post-surgical osteolysis.
Additionally, the present invention is directed to a method for selecting an agent for treating a disorder mediated by a matrix metalloproteinase. The method comprises selecting an agent which increases endocytosis of the matrix metalloproteinase.
Among the several advantages achieved by the present invention, therefore, may be noted the provision of methods for inactivating matrix metalloproteinases by causing an increase in endocytosis and degradation of the enzymes; the provision of methods for increasing endocytosis of collagenase-3 through treatment with an agent which increases collagenase-3 receptor activity; the provision of methods for treating disorders mediated by matrix metalloproteinase activity, where the methods effect an increase in endocytosis of the matrix metalloproteinase; and the provision of methods for determining whether an agent is effective in treating a disorder mediated by a matrix metalloproteinase, where the methods test for an increase in endocytosis of the matrix metalloproteinase.